Currently, there are two ways to determine the amount of sialic acid of IgG immunoglobulin G: the first one was published 1997 in “The journal of Immunology vol. 159, p. 2327-2333,” titled “The Glycosylation of IgA produced by Murine B cells is altered by Th2 cytokines.” The steps are as following: Step 1: in enzyme-linked immunosorbent assay (ELISA) plates, adding IgG anti mouse IgG (Fab′) 2 fragments into each well; using 0.1% bovine serum albumin in phosphate buffered saline (PBS) washing three times; and adding 1% bovine serum albumin in PBS to prevent non-specific binding; Step 2: after washing three more times, adding different concentrations of IgA obtained from CH12LX cell separation for two hours; Step 3: adding 5 μg/ml of biotinylated Sambucus nigra agglutinin for ninety minutes; Step 4: after washing three more times, adding alkaline phosphate linked avidin (streptavidin) (1 μg/ml) for ninety minutes; and Step 5: finally washing three more times, adding chromogenic substance P-nitrophenyl phosphate disodium, and measuring the absorption value at 405 nm in an enzyme-linked immunosorbent assay (ELISA) reader. This method is disadvantageous because it cannot use culture fluid, blood, plasma, or serum to directly measure the amount of sialic acid in different immunoglobulins (IgG, IgA, IgM).
Another method to measure the sialic acid of immunoglobulin G uses the immunoassay of lectin inhibiting enzyme, published in “The Journal of PLOS ONE, 2011, vol. 6: e21246,” titled “Enrichment of sialylated IgG by lectin Fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia.” The steps are as following: Step 1: diluting biotinylated Sambucus nigra agglutinin with 0.1% polyoxyethylene (20) sorbitan monolaurate (Tween 20) in PBS to 0.5 μg/ml, the solution mixing with the inhibitor (intravenous immunoglobulin; intravenous immunoglobulin and enriched sialylated IgG; intravenous immunoglobulin containing only desialylated immunoglobulin; and immunoglobulin Fab or immunoglobulin Fc fragments) in a test tube at room temperature for one hour; Step 2: adding intravenous immunoglobulin to an ELISA plate; Step 3: adding the mixture of Step 1 above to the ELISA plate at room temperature for 1 hour; Step 4: After washing, adding streptavidin-linked horseradish peroxidase (at 1:4000 dilution) with 0.1% Tween 20/phosphate buffered saline solution, and incubating the plate at room temperature for one hour; Step 5: After washing, adding 100 μl of tetramethyl benzidine (TMB) substrate buffer (Interchim, Montlucon, Cedex, France) and distilled water into each well, and adding 2 molar (M) concentration of sulfuric acid (H2SO4) to stop the reaction after 5 minutes; and Step 6: measuring the absorption value at 450 nm in the enzyme-linked immunosorbent assay (ELISA) reader. This method is disadvantageous because there is no sialic acid standard available and it cannot directly measure the amount of sialic acid in different immunoglobulins (IgG, IgA, or IgM).
And in academia or industry, there is no publications related to the measurement of the amount of sialic acid in IgG anti-double-stranded DNA. Therefore, there remains a need for a new and improved method to measure the amount of sialic acid in IgG anti-double-stranded DNA.